DNA microarray analysis of the anti-inflammatory effects of PDTC on IL-6 signaling in HepG2 cells

Interleukin-6 (IL-6) is a proinflammatory cytokine that exerts a wide range of cellular, physiological and pathophysiological responses. Pyrrolidine dithiocarbamate (PDTC) antagonizes the cellular responsiveness to IL-6 through impairment in STAT3 activation and downstream signaling. Here, a transcriptional profiling was conducted as a basis for understanding the biological properties of PDTC in human HepG2 hepatocarcinoma cells. A global comparison of mRNA identified a highly significant difference of dysregulated gene expression transduced by PDTC versus IL-6 in HepG2 cells. Through an unbiased pathway analysis method, we have uncovered the mammalian target of rapamycin (mTOR) pathway together with rapid and dynamic alterations in REDD1 (regulated in development and DNA damage response 1) expression as one of the underlying molecular mechanisms responsible for IL-6 resistance to PDTC. Quantitative PCR and Western blot analyses validated the microarray data by showing the reciprocal pattern of REDD1 expression and subsequent mTOR inhibition after stimulation with PDTC relative to IL-6. The HepG2 human hepatocarcinoma cell line (American Type Culture Collection) was routinely grown and maintained in Minimum Eagle's medium (MEM)/F12 (1:1) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50 ug/ml streptomycin under standard cell culture conditions at 37°C and 5% CO2 in a humid environment. Cells were seeded in 35- or 60-mm dishes and grown to ~90% confluence after which they were serum starved for 4 h followed by the addition of either vehicle, PDTC (50 µM) or human recombinant IL-6 (20 ng/ml) for 2 replicates, labeled a and b, of the 5 time points: 0hr, 1hr, 2hr, 4hr and 8hr.